Synthetic peptide lung surfactants having covalently bonded antioxidants

ABSTRACT

Synthetic pulmonary surfactants having antioxidant properties consisting of a complex of a polypeptide of 3-4 amino acid residues, with an antioxidant moiety, and a lipid consisting of one or more of the lipids associated with natural pulmonary surfactant were prepared. These surfactants are useful in the treatment of respiratory distress syndrome.

CROSS-REFERENCE TO RELATED APPLICATION

This is a division of application Ser. No. 08/502,722, filed Jul. 14,1995, now U.S. Pat. No. 5,623,052; which is a continuation ofapplication Ser. No. 08/077,802, filed Jun. 21, 1993; now abandonedwhich is a continuation-in-part of application Ser. No. 07/923,092,filed Jul. 31, 1992, now abandoned which is herein incorporated byreference.

FIELD OF THE INVENTION

This invention relates to the synthesis of a series of 3 to 4 amino acidpolypeptides having antioxidants covalently linked to the peptide eitherdirectly or through a linker region. These modified peptides are usefulas synthetic lung surfactants having useful antioxidants structurally aspart of the peptide. Also described are the preparation of mixtures ofthese polypeptides with lipids, the method for production of same, andpharmaceutical compositions which are effective in the treatment ofmammalian respiratory distress syndromes.

BACKGROUND OF THE INVENTION

The lungs exist in a delicate balance between toxic oxidants and theprotective activities of antioxidant defense systems. An imbalance inthis system, either through an increase in oxidants or a dysfunction ofthe protective antioxidant defense systems, can lead topathophysiological events in the lung causing pulmonary dysfunction. Onetype of pulmonary dysfunction in which an increase in oxidants cancontribute is respiratory distress syndrome (RDS).

Infantile respiratory distress syndrome is a leading cause of death inthe first 28 days of life. Infantile RDS strikes 1 in 100 babiesworldwide and about 10 percent die. The syndrome rarely occurs in terminfants but is generally associated with immaturity and low-birth weight(under 2 kg). Adult RDS shows similar clinical characteristics andpathophysiology to the infantile disease and is generally managed in anintensive care facility. The adult disease has diverse etiologies, manyresulting from lung insults, such as diffuse infections, aspiration ofthe gastric contents, inhalation of irritants and toxins, and pulmonaryedema arising from such sources as narcotic overdose.

RDS is correlated with an absence or dysfunction of the lung surfactantwhich coats the alveoli of the lungs where gas exchange occurs, and hasbeen associated with oxygen centered free radicals in the lung or lungcavity known as oxidants, such as superoxide radicals, hydroxylradicals, hydrogen peroxide which can generate hydroxyl radicals, andlipid peroxides, which have been implicated in cellular injury (Heffner,et al., Am. Rev. Respir. Dis. 104: 531-554 1989); (Halliwell, FASEB J.1: 358-364 1987).

Synthetic lung surfactant of larger polypeptides having antioxidantmoieties, have been described in U.S. Pat. application Ser. No.07/789,918 filed filed Nov. 4, 1991, which is herein incorporated byreference. However, the present invention provides an effectivesynthetic lung surfactant having antioxidant properties to shortenedpeptides of 3-4 amino acids having the ability to inhibit oxidation ofsusceptible compounds into oxidants. The shortened lung surfactantsprovide a more efficient and more cost effective means of producingtherapeutics. The present novelty of the invention resides in theability to effectively reduce the peptide to 3-4 amino acids with theretention of surfactant properties and effectively deliver the peptideattached to a covalently bonded antioxidant.

Some synthetic lung surfactant preparations have added therapeuticagents such as Vitamin E to surfactant preparations as a separatecomponent (U.S. Pat. No. 4,765,987; PCT Publication No. WO 90/11768; PCTpublication no. WO 90/07469). However, in the present invention theantioxidants are not a separate component but are actually incorporatedinto a polypeptide. An advantage of incorporating the antioxidant intothe polypeptide is that instead of having a three component mixture(lipid, polypeptide and antioxidant), a two component mixture isavailable. This can be a significant advantage in testing for efficacyfor a marketable pharmaceutical where a variety of dosages andformulations must be tested for each component. Additionally, a twocomponent formulation is easier to manufacture.

The polypeptides of the present invention may be used singly in mixtureswith lipid or in combination in mixtures of lipid wherein thepolypeptide comprises a minor component of the surfactant mixture. Thecomposition of the present invention may be prepared in high purity andin a standardized fashion as it is a defined mixture of syntheticcomponents. Also, the components are not derived from animal sourceswhich minimizes the risk of contamination by viruses and bacteria.

A helical wheel representation of an amphipathic α-helical ten-residuepeptide (for description of the amphipathic α-helical peptide seeMcLean, L. R. et al. Biochem., 1991, 30, 31) is used to develop a modelfor three and four residue peptides. When looking down the barrel of theα-helix, the side chains of the residues indicate a hydrophobic face anda hydrophilic face on the helix. A four residue peptide represents asingle turn of this α-helix with the required hydrophobic andhydrophilic face present. A three residue peptide represents aconstricted turn of the α-helix with the hydrophobic and hydrophilicface still present.

SUMMARY OF THE INVENTION

The present invention comprises synthetic lung surfactants consisting ofa complex of a polypeptide and lipids wherein the polypeptide has thefollowing formula 1:

    X--A.sub.1 --A.sub.2 --A.sub.3 --A.sub.4 --Y               1

or an optically active isomer or pharmaceutically acceptable saltthereof; wherein

A₁ is a bond or negatively charged amino acid selected from Glu or Asp;

A₂ is a hydrophobic amino acid selected from Trp, Tyr, Phe, His, Val,Leu, or Ile;

A₃ is Aib, Glu, Gln, Leu, Ala, Orn or a bond; and

A₄ is a positive charged amino acid selected from Lys, Arg, or His;

X is of formula Da or Db: ##STR1## wherein, B₁ is B, --C(O)--,--B--C(O)--, --C(O)--NH--B--C(O)--; and B is a bond, C₁₋₁₆ alkylene, orC₂₋₁₆ alkenylene; and wherein each R₁, R₂, R₃, R₄, R₅, R₆ and R₇ isindependently a C₁₋₆ alkyl;

Y is a carboxyl substituent of A₄ selected from hydroxy, amino,alkylamino, and alkoxy groups; and

wherein, when A₃ is a bond, A₁ and A₂ may be interchanged.

In addition the present invention comprises synthetic lung surfactantsconsisting of a complex of a polypeptide and lipids wherein thepolypeptide has the following formula 2:

    X--A.sub.1 --A.sub.2 --A.sub.3 --A.sub.4 --Y;              2

or an optically active isomer or pharmaceutically acceptable saltthereof; wherein

A₁ is a bond or Glu;

A₂ is Trp or Glu;

A₃ is Aib, Glu, Gln, Leu, Ala or Orn; and

A₄ is Lys;

X is of formula Da or Db: ##STR2## wherein B₁ is B, --C(O)--,--B--C(O)--, --C(O)--NH--B--C(O)--; and B is a bond, C₁₋₁₆ alkylene, orC₂₋₁₆ alkenylene; and wherein each R₁, R₂, R₃, R₄, R₅, R₆ and R₇ isindependently a C₁₋₆ alkyl; and

Y is a carboxyl substituent of A₄ selected from hydroxy, amino,alkylamino, and alkoxy groups.

Further the peptides of this invention may be associated with a lipid,comprised of one or more of the type associated with natural pulmonarysurfactant.

These polypeptide-lipid complexes and their pharmaceutical compositionsare useful in treating mammalian respiratory distress syndrome.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a helical wheel representation of a ten-residue peptidesurfactant used to develop a model for short peptides. The view is downthe barrel of the helix and the side chains of the residues areindicated in their positions relative to the axis of the helix. Thehydrophobic face includes the residues to the right in the drawing whichare Trp⁸, Leu¹, Leu⁵, Leu⁹, Leu² and Leu⁶. The hydrophilic face includesthe charged residues Lys⁴, Glu⁷, Glu³ and Lys¹⁰.

FIG. 2 is an example of a tetrapeptide antioxidant designed on the basisof a single turn of the helical wheel projection of the ten-residuepeptide shown in FIG. 1. The hydrophobic face of the FIG. 1 peptide hasbeen replaced by Trp², Ala³, HBB-Aoc which present a sufficienthydrophobic face to anchor the peptide to the lipid. The hydrophiliccharged face has been replaced by Glu¹ and Lys⁴.

BRIEF DESCRIPTION OF TABLES

Table I shows the results from amino acid analysis of the synthesizedpeptides.

Table II shows the results of pressure-volume experiments showing theeffectiveness of compounds in the adult rat lung model.

DETAILED DESCRIPTION OF THE INVENTION

The following common abbreviations of the naturally occurring aminoacids are used throughout this specification:

Ala or A--alanine

Val or V--valine

Leu or L--leucine

Ile or I--isoleucine

Phe or F--phenylalanine

Trp or W--tryptophan

Met or M--methionine

Ser or S--serine

Tyr or Y--tyrosine

Asp or D--aspartic acid

Glu or E--glutamic acid

Gln or Q--glutamine

Thr or T--threonine

Gly or G--glycine

Lys or K--lysine

Arg or R--arginine

Asn or N--asparagine

Nle--norleucine

Orn--ornithine

hArg--homoarginine

Nva--norvaline

Aib--amino-isobutyric acid

The natural amino acids, with the exception of glycine, contain a chiralcarbon atom. Unless otherwise specifically indicated, the opticallyactive amino acids, referred to herein, are of the L-configuration. Oncethe antioxidant moiety of the present invention is added to the peptide,stereoisomers can be formed. The present invention comprises mixtures ofsuch stereoisomers as well as the isolated stereoisomer. As iscustomary, the structure of peptides written out herein is such that theamino terminal end is on the left side of the chain and the carboxyterminal end is on the right side of the chain.

When two amino acids combine to form a peptide though a typical amidebond, a molecule of water is released, and what remains of each aminoacid is called a "residue". The amide linkage can also occur when X islinked to a subsequent amino acid or to an amide bond isoster. A residueis therefore an amino acid that lacks a hydrogen atom of the terminalamino group, and lacks the hydroxyl group of the terminal carboxylgroup. Using accepted terminology, a dash (--) in front of (indicatingloss of a water) a three letter code for an amino acid or amino acidderivative indicates the amine bond of a residue.

"Alkyl" as used herein means a straight or branched chain hydrocarbonradical such as methyl, ethyl, propyl, butyl, isopropyl, tert-butyl,sec-butyl, isopentyl, l-methybutyl and so on, depending upon the numberof carbon atoms specified. "Acyl" as used herein means a radical formedfrom an organic acid by removal of a hydroxyl group; the general formulais RCO-- where R may be aliphatic, alicyclic, aromatic hydrocarbon orhydrogen (formyl group). The R group may be substituted. An example ofan acyl group is succinyl.

As used herein the term "hydrophobic amino acid" means a nonpolarresidue with an aliphatic hydrocarbon side chain such as Val, Leu orIle; or a nonpolar residue with an aromatic group such as Phe, Tyr, Trpor His.

As used herein the term "negatively charged amino acid" means a polarresidue with an acidic hydrophilic side chain such as Glu or Asp.

As used herein the term "positive charged amino acid" means a polarresidue with a basic hydrophilic side chain such as Lys, Arg or His.

Peptides, where X has not been functionally modified by the designatedantioxidant, can be synthesized by any suitable method such as solidphase sequential procedure, described hereafter. Preferred Markushgroups are where, R₁, R₂, R₆ and R₇ are each tert-butyl, and each of R₃,R₄ and R₅ are methyl. Da is preferable to Db, and B is preferably--C(O)--NH--B--C(O)--, wherein B is a C₈ alkane;

X is referred to herein as "antioxidant moiety" because it is believedthat X is that portion which confers antioxidant properties on thepolypeptide. However, it is to be understood that X may have linkers tothe polypeptide so that when antioxidant moieties attached to thepolypeptide are described, it also includes the appropriate linkers,e.g., B, --C(O)--, B--C(O)--, C(O)--NH--B--C(O)--, etc.

There are many ways to form X. For example, amino acid derivatives canbe acylated by an acylating agent formed from antioxidant compounds. Tobe an acylating agent, the antioxidant compounds can, for example, forma symmetrical anhydride or an active ester, e.g., N-hydroxybenzotriazoleester (HOBt ester). The acylating agent is then exposed to theunprotected functional nucleophile for the reaction to take place. Thisis preferably performed in solid phase peptide synthesis while the aminoacid to receive the antioxidant moiety is part of the peptide attachedto the resin.

Individual amino acids can also be modified prior to incorporation intothe peptide by, for example, esterification, reductive alkylation, etc.Other modifications of amino acids and amino acid derivatives containingfunctional groups are well known in the art.

Preferred examples of antioxidant compounds found to be useful inreacting with amino acids or amino acid derivatives in the presentinvention are as follows:

1) HBB=3,5-di-t-butyl-4-hydroxybenzoic acid

2) HBP=3-(3',5'-di-tert-butyl-4-hydroxyphenyl)-propionic acid

3) HBC=3,5-di-tert-butyl-4-hydroxycinnamic acid

4) HBA=2-(3',5'-di-t-butyl-4-hydroxyphenyl) acetic acid

5) di-HBA=2,2-di-(3',5'-di-t-butyl-4-hydroxyphenyl)-acetic acid

6) Trl=6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid -alsoknown as Trolox

Preferably HBB, HBP, HBC, HBA, di-HBA, and Trl are used when thefunctional group is either an alcohol group or an amino group. Withinthe group of linked surfactants a preferred grouping can be selected toform a more preferred grouping, such as, HBB and Trl.

The foregoing antioxidant compounds are commercially available or thesynthesis known in the art, e.g., 3,5-di-t-butyl-4-hydroxyphenylaceticacid is described in Izv. Akad. Nauk SSSR, Ser. Khim., 358 1965 and3,5-di-t-butyl-4-hydroxy-benzaldehyde is described in J. Org. Chem., 22,1333 1957. Generally, any antioxidant compound may be used in thepresent invention which (1) can be attached to the polypeptide of thepresent invention, (2) exhibits antioxidant activity while attached tothe polypeptide, and (3) permits the polypeptide to perform as describedherein.

Trl-Glu--means a molecule having a peptide bond formed between troloxand a glutamyl residue; wherein the trolox is attached to the α-aminogroup of a Glutamic acid residue as shown below: ##STR3##

As shown by the Trl-Glu example, the antioxidant moiety, in this casewhere X=Db and B=a bond, together with a carbonyl group (C(O)--) can beattached to the α-amino terminus of polypeptide to form Db--C(O)--A₁--A₂ --A₃ --A₄ --Y.

The polypeptides of this invention can be prepared by a variety ofprocedures readily known to those skilled in the art such as solutionphase chemistry. A preferred method is the solid phase sequentialprocedure which can use automated methods such as the ABI peptidesynthesizer. In solid phase sequential procedure, the following stepsoccur: (1) a first amino acid, having a protected α-amino group, isbound to a resin support; (2) the carboxylic group of a second aminoacid, having a protected α-amino group, is activated; (3) the firstamino acid is deprotected with a reagent which permits the first aminoacid to remain attached to the resin; and (4) coupling occurs betweenthe α-amino group of the first amino acid and the activated carboxylicgroup of the second amino acid. These steps are repeated with new aminoacid residues which permits the formation of the peptide. When thedesired length of peptide has been formed, the peptide may be modifiedwith an appropriately coupled antioxidant moiety prior to being cleavedfrom the resin, deprotected and isolated. Alternatively the protectedpeptide may be selectively removed from the resin, and the antioxidantmoiety is coupled to the peptide prior to removal of protecting groupsand isolation.

The resin support employed can be any suitable resin conventionallyemployed in the art for the solid phase preparation of polypeptides suchas a polystyrene which has been cross-linked with from 0.5 to about 3percent divinyl benzene, which has been either chloromethylated orhydroxymethylated to provide sites for ester formation with theinitially introduced α-amino protected amino acid. Other suitable resinsupports are pMHBA (Peptide International, Louisville, Ky.), RINK(Calbiochem, LaJolla, Calif.) and Sasrin (Biochem, Philadelphia, Pa.).The Sasrin resin requires a special ABI cycle for loading the firstamino acid which is described in the ABI peptide synthesizer user'smanual. The first amino acid, having a protected α-amino group, isattached to the resin as described in the Applied Biosystems Model 430APeptide Synthesizer User's Manual, incorporated in its entirety herein.

Preferred methods of activating each added amino acid to the boundpeptide chain include formation of a symmetrical anhydride or activeester of the each added α-amino which has been appropriately protected.For example, an α-amino protected amino acid can be reacted withdicyclohexylcarbodiimide (DCC) in the presence of dichloromethane (DCM)to form the symmetrical anhydride. Alternatively, a HOBt active estercan be formed by dissolving Boc-amino acid (tert-butyloxycarbonyl-aminoacid) and HOBt in DCC and chilling, adding additional DCC and warmingthe solution to room temperature. This solution is then added to theamino acid bound resin. This method of activation to form acylatingagents may also be used for the antioxidant compounds.

If there are other functional groups present besides the α-amino group,those groups will generally have to be protected. Generally, the α-aminogroup and each of the side chain functional groups can be protected bydifferent protecting groups so that one protecting group can be removedwithout removing the other protecting groups.

Among the classes of α-amino protecting groups contemplated for use withthe present invention are (1) acyl type protecting groups such as:formyl, trifluoroacetyl, phthalyl, toluenesulfonyl (tosyl),benzenesulfonyl, nitrophenylsulfenyl, tritylsulfenyl,o-nitrophenoxyacetyl and γ-chlorobutyryl; (2) aromatic urethan typeprotecting groups such as benzyloxycarbonyl and substitutedbenzyloxycarbonyl such as p-chlorobenzyloxycarbonyl,p-nitrobenzyloxycarbonyl, p-bromobenzyloxycarbonylbromobenzyloxycarbonyl, p-methoxybenzyloxycarbonyl,l-(p-biphenyl)-l-methylethoxycarbonyl,α,α-dimethyl-3,5-dimethoxybenzyloxycarbonyl and benzylhydryloxycarbonyl;(3) aliphatic urethan protecting groups such as tert-butyloxycarbonyl(Boc), diisopropylmethoxycarbonyl, isopropyloxycarbonyl, ethoxycarbonyland allyloxycarbonyl; (4) cycloalkyl urethan type protecting groups suchas cyclopentyloxycarbonyl or 9-fluorenylmethoxycarbonyl (Fmoc); (6)alkyl type protecting groups such as triphenylmethyl (trityl) andbenzyl; (7) trialkylsilane groups such as trimethylsilane.

The selection of the α-amino protecting group, however, will depend uponthe resin used, the target site functional group, the other functionalgroups present in the polypeptide and whether the amino acid derivativeX can withstand cleavage from the resin with the cleavage reagent. Forexample, to prepare HBB-Aoc-Glu-Trp-Aib-Lys-NH₂, (SEQ ID NO: 1), a pMBHAresin is used, which produces a C terminal amino group, and the peptideis constructed using standard t-Boc chemistry on an ABI430A peptidesynthesizer. The HBB moiety can be introduced as an HOBT active ester inorder to attach HBB at the target site N-α-amino group of Glutamic acid.Anhydrous hydrofluoric acid (HF) can be used to simultaneously cleavethe peptide from the resin and to remove the remaining protectinggroups.

The selection of appropriate combination of protecting groups andreagents to selectively remove protecting groups is well known in theart. For example, see M. Bodanszky, PEPTIDE CHEMISTRY, A PRACTICALTEXTBOOK, Springer-Verlag (1988); J. Stewart, et al., SOLID PHASEPEPTIDE SYNTHESIS, 2nd ed., Pierce Chemical Co. (1984).

Each protected amino acid or amino acid sequence is introduced into thesolid phase reactor in about a four-fold excess and the coupling iscarried out in the presence of a coupling agent such as in a medium ofdimethylform-amide: methylene chloride (1:1) or in dimethylformamidealone or methylene chloride alone. In cases where incomplete couplingoccurs, the coupling procedure is repeated before removal of the α-aminoprotecting group, prior to the coupling of the next amino acid in thesolid phase reactor. The success of the coupling reaction at each stageof the synthesis is monitored by the ninhydrin reaction as described byE. Kaiser, et al., Analyt. Biochem. 34, 595 (1970).

After the desired amino acid sequence has been obtained, the peptide isremoved from the resin using any appropriate reagent which will notadversely effect the polypeptide. For example, anyhdrous HF containing5% anisole and 5% acetonitrile in 0.1% trifluoroacetic acid can be usedto cleave the polypeptide from a pMBHA resin.

The polypeptides of Formula 1 can form pharmaceutically acceptable saltswith any non-toxic, organic or inorganic acid. Illustrative inorganicacids which form suitable salts include hydrochloric, hydrobromic,sulphuric and phosphoric acid and acid metal salts such as sodiummonohydrogen orthophosphate and potassium hydrogen sulfate. Illustrativeorganic acids which form suitable salts include the mono, di andtricarboxylic acids. Illustrative of such acids are, for example,acetic, glycolic, lactic, pyruvic, malonic, succinic, glutaric, fumaric,malic, tartaric, citric, ascorbic, maleic, hydroxymaleic, benzoic,hydroxybenzoic, phenylacetic, cinnamic, salicylic, 2-phenoxbenzoic andsulfonic acids such as methane sulfonic acid and 2-hydroxyethanesulfonic acid. Salts of the carboxy terminal amino acid moiety includethe non-toxic carboxylic acid salts formed with any suitable inorganicor organic bases. Illustratively, these salts include those of alkalimetals, as for example, sodium and potassium; alkaline earth metals,such as calcium and magnesium; light metals of Group IIIA includingaluminum; and organic primary, secondary and tertiary amines, as forexample, trialkylamines, including triethylamine, procaine,dibenzylamine, 1-ethenamine, N,N'-dibenzylethylenediamine,dihydroabietylamine, N-(lower)alkylpiperidine, and any other suitableamine.

The phospholipids of the protein-phospholipid complexes of thisinvention can be any phospholipid and this term as used herein includesthe phosphoglycerides and the sphingolipids. Phosphoglycerides are thosedi-fatty acid esters of glycerol in which the remaining hydroxy group, aterminal hydroxy group, of the gylcerol moiety forms an ester withphosphoric acid. Commonly the phosphoric acid moiety of thephosphoglycerides forms a second ester with an alcohol such asethanolamine, serine, choline, or glycerol. Sphingolipids are thosemono-fatty acid esters of sphingosine or dihydrosphingosine in which thehydroxy group at the 1-position forms an ester with the choline ester ofphosphoric acid. The preferred lipids of the protein-phospholipidcomplexes of this invention comprise dipalmitoylphosphatidylcholine(DPPC), phosphatidylcholine molecules containing acyl chains of otherlengths and degrees of saturation (PC), cardiolipin (CL),phosphatidylglycerols (PG), phosphatidylserines (PS), fatty acids (FA),and triacylglycerols (TG). DPPC comprises the major component of thelung surfactant mixture while PC, CL, PG, PS, FA, and TG comprise minorcomponents. Suitable fatty acids for use in the phospholipids of thisinvention are long chain carboxylic acids (generally having eight ormore carbon atoms), typically unbranched. The fatty acids can be eithersaturated or unsaturated. Representative fatty acids are lauric,myristic, palmitic, and oleic acids.

Pharmaceutical preparations of the polypeptide or theprotein-phospholipid complexes of this invention can be prepared as adry mixture or in an aqueous suspension, in some instances containingsmall amounts of organic solvents, such as, for example, ethanol ortrifluoro-ethanol, detergents, such as, for example, sodium dodecylsulfate or sodium deoxycholate, salts, such as calcium chloride orsodium chloride, carbohydrates, such as glucose, dextrose or mannitol,and amino acids, such as glycine and alanine. Where the pharmaceuticalcomposition is made into liquid form, stabilizers, preservatives,osmotic pressure regulators, buffering agents, and suspending agents ofthe liquid may be added. If desired, suitable germicides may also beadded. The pH of the aqueous suspension may vary between 2 and 10 andmay be adjusted with acids and bases, such as, for example, hydrochloricacid, sodium phosphate, or sodium hydroxide. The dry mixture may bereconstituted in an aqueous solution containing pharmaceuticallyacceptable salts, organic solvents, and detergents. The aqueouspreparation may be dialyzed, filtered, or chromatographed to exchangethe suspending medium with a pharmaceutically acceptable medium prior touse. The preparation may be administered as a dry powder, an aqueoussuspension, or as an aerosol directly into the lungs of the distressedsubject. The pharmaceutical composition of the present invention may becharged in hermetically sealed containers such as vials and ampules andbe preserved sterilely. The composition may be stored in a vial orampule separately from a vial or ampule containing the suspension bufferand the dry or hydrated composition may be mixed with the suspensionbuffer prior to use.

Lipid constitutes from 50 to 99.9% of the lung surfactant preparation.Suitable lipids include DPPC, PC, CL, PG, PS, FA, and TG. DPPC comprisesthe major lipid species and is present in concentrations of 60 to 100%of the total lipid weight. The remaining lipids are present in minorconcentrations. PC, CL, PG and PS may comprise up to 30% of the lipids,and FA and TG may comprise up to 10% of the lipid weight. The fatty acylchains of the minor lipid components may be saturated or unsaturated andof any chain length. Chain lengths of 12 to 16 carbon atoms and up to 2unsaturated bonds are preferred. The preferred lipid composition is85-100% DPPC plus 0-15% of PG. Most preferred is pure DPPC.

The lipid components of the synthetic lung surfactant are commonly foundin mammalian lung surfactant and are available from common industrialsources in high purity. The polypeptide components are prepared bysolid-phase peptide synthesis by methods familiar to those skilled inthe art. Mixtures of the lipids of the invention with proteins isolatedfrom mammalian lung lavage have been shown to be effective in treatingneonatal RDS. However, mixtures of these lipids with synthetic peptidesin lung surfactant preparations has only recently been reported (McLean,et al.).

Lipids are suspended as liposomes by methods familiar to those skilledin the art; i.e., wherein initially lipids are mixed in a volatileorganic solvent or mixtures of solvents, such as mixtures of chloroformand methanol or trifluoroethanol. The organic solvent is removed byevaporation under nitrogen, argon, or under vacuum. An aqueous solutionwhich may contain organic and inorganic acids, bases, and salts, andsaccharides such as dextrose is added to the dry lipid mixture to attaina final concentration of 0.1 to 100 mg of DPPC per ml. In general, it ispreferable, but not necessary to warm the mixture to 35°-50° C., mixvigorously, and incubate for up to 2 hours at 25°-50° C. Then, pepcideor a mixture of peptides is added as a dry powder or suspended in anaqueous solution in some cases containing a suitable organic solvent,such as ethanol or trifluorethanol, or a denaturing agent, such asguanidinium hydrochloride or urea, which improves the solubility of thepeptide in the aqueous suspension. Association of peptide and lipid maybe promoted at a particular pH, thus the pH of the aqueous solution mayvary from 2 to 10. The preferred method for mixing peptide and lipid isto add dry peptide to lipid in water at 45°-50° C. and to mix by bathultrasonication at 45°-50° C. for 30-90 minutes, then freeze-dry andstore at -20° C.

Lipids can optionally be mixed with a suitable detergent such asoctylglucoside or sodium deoxycholate at a weight ratio of from 1 to 20parts of detergent per part of DPPC in water, an aqueous buffer, orsaline solution at concentrations from 1 to 100 mg DPPC/ml. Then,peptide is added as a dry powder or suspended in an aqueous solutionwith or without an organic solvent, denaturing agent, or detergent. Themixture is then dialyzed, filtered, centrifuged or chromatographed toremove the detergent.

Preferably, lipids and peptides are mixed in a volatile organic solventwith or without a small amount of water. The volatile solvent isevaporated under a stream of nitrogen or argon, in a vacuum oven, or byrotary evaporation either before or after addition of an aqueoussolvent.

The mixture of lipid and peptide prepared by one of the methodsdescribed above is incubated for up to 2 hours, preferably at 35°-50° C.with sonic irradiation. The mixture may then be dialyzed, filtered, orchromatographed to replace the aqueous medium with a pharmaceuticallyacceptable medium, although this is not necessary. In some cases,efficacy is improved by separating unreacted lipid or peptide fromassociated lipid and peptide by ultracentrifugation, filtration, orchromatography. The mixture may then be lyophilized or aerosolized.

When the polypeptide-phospholipid complexes of this invention are usedin the treatment of neonatal respiratory distress syndrome, aphysiological condition which results from the inability of the lungs ofpremature infants to produce pulmonary surfactant, the complexes act asan antioxidant and synthetic pulmonary surfactants and either replacethe natural, missing surfactant or augment the lack of sufficientnatural surfactant. Treatment is continued until the infant's lungsproduce a sufficient amount of natural, pulmonary surfactant so as torender further treatment unnecessary.

The preparations are preferably those suitable for endotrachealadministration, that is as a liquid suspension, a dry powder, or anaerosol. For a liquid suspension, the dry mixture or the mixture inaqueous suspension is mixed with suitable agents, such as water, salinesolutions, dextrose, and glycerol to produce a pharmaceuticallyeffective composition. Preferred liquid suspensions will contain 0.8 to1.0 weight per cent of sodium chloride and will be 1-20 mM, preferablyin calcium ion. The preparation is then filter sterilized. In general,the preparation comprises 1 to 100 mg of DPPC per ml and is administeredat a dose of 0.2 to 5 ml/kg. To prepare a dry mixture, the aqueoussuspension is lyophilized. The aerosol is prepared from a finely divideddry powder suspended in a propellant, such as lower alkanes andfluorinated alkanes, such as Freon. The aerosol is stored in apressurized container.

For example, the surfactant (polypeptide of the present invention andlipid complex) is administered, as appropriate to the dosage form, byendotracheal tube, by aerosol administration, or by nebulization of thesuspension or dry mixture into the inspired gas. The surfactant isadministered in one or multiple doses of 10 to 200 mg/kg. The preferredmethod of administration is as a suspension of peptide and lipid inphysiological saline solution at a concentration of 5-10 mg ofsurfactant per ml through an endotracheal tube, achieving a dose of50-100 mg/kg.

The polypeptide of the present invention is administered to treat asubject. "Subject" means a mammal, for example, but not limited to, ahuman being.

The following examples show some methods of preparation for thepolypeptide, polypeptide/lipid complex and starting materials of thepresent invention. The present invention is not limited to the followingexamples nor to these methods of preparation.

Abbreviations used in the examples not previously defined are asfollows:

TBDMS Tetrabutyldimethylsilyl

SEt Ethylthio

Suc Succinyl

TFA Trifluoroacetic acid

Bzl Benzyl

Ot-Bu t-butyl ether;

which accompany standard Boc chemistry and standard Fmoc chemistry: thatchemistry used with the ABI peptide synthesizer respectively for the Boccycles and the Fmoc cycles.

EXPERIMENTAL CHEMICAL PROCEDURES EXAMPLE 1

Peptide Synthesis and other Chemicals. Peptides were synthesized on a0.5 mmol scale by solid-phase methods on an Applied Biosystems Inc.(Foster City, Calif.) Model 430-A peptide synthesizer.p-methylbenzoxyhydrylamine (pMBHA) resin was used to give C-terminalamides on cleavage. Nα-t-Boc (t-butyloxycarbonyl) amino acids with sidechain protection Cys(ethylthio), Clu(benzyl) andLys(2-chlorobenzyloxycarbonyl) from Peptides International weredouble-coupled via their preformed symmetrical anhydrides. Theantioxidant group, was coupled by activating the acid of the antioxidantto form the symmetrical anhydride. Antioxidants, such as HBB (3,5,di-tert-butyl-4-hydroxy benzoic acid) were placed at the amino terminusof the peptide by preactivating the HBB acid to form the correspondingsymmetrical anhydride. Generally the antioxidant was double or triplecoupled to assure complete reaction. For example HBB required threecouplings to achieve complete incorporation. Additional couplings wereperformed as determined based on ninhydrin tests. Nα-t-Boc groups wereremoved with 50% trifluoroacetic acid (TFA) in methylene chloride andneutralized with 10% diisopropylethylamine (DEA) in dimethyl formamide.The peptides were cleaved from the resin and deprotected in anhydrous HFcontaining 5% anisole and 5% dimethyl sulfide at -5° C. for 45 min. HFwas removed in vacuo and the peptide extracted from the resin with 50%aqueous acetonitrile. The combined extracts were frozen and lyophilizedand purified by reverse phase preparative HPLC on a Rainin Dynamax(21.4×250 mm) C₁₈ column at 40 mL/min with an acetonitrile gradient in0.1% aqueous TFA (pH 2) monitored at 214 nm. The major peak wascollected and lyophilized. The purity (>97%) and identity of thesynthetic peptides were confirmed by a single peak in the analyticalhigh performance liquid chromatogram (HPLC), capillary zoneelectrophoresis, fast-atom bombardment mass spectrometry (FAB-MS) on aVG Analytical ZAB2-SE which gave single molecular ions consistent withthe correct sequences, and amino acid analyses which were within 10% ofthe predicted values for each residue.L-α-dipalmitoylphosphatidylcholine (DPPC) (>99% pure) was from AvantiPolar Lipids (Birmingham, Ala.). Using these procedures the followingpeptides were synthesized; their analytical properties are found inTable 1.

1(A). Preparation of Polypeptide HBB-Aoc-Glu-Trp-Aib-Lys-NH₂ (SEQ IDNO: 1) (HBB-Aoc=N.sup.α -hydroxy-di-t-butyl-benzoyl-aminoocatanoyl-)

Initially Aoc-Glu(OBzl)-Trp-Aib-Lys(N.sup.ε -2ClZ)-pMBHA was prepared byusing a Lys(N.sup.ε -2ClZ)-pMBHA resin placed in ABI430A peptidesynthesizer and synthesized using standard t-Boc chemistry. Tosynthesize peptide 1A, N.sup.α -hydroxy-di-t-butyl-benzoic acid (HBB)(501 mg), dimethylformamide (4 mL) and methylene chloride (4 mL) werecombined and a dicyclohexylcarbodiimide solution (8 mL of a 0.5Msolution in methylene chloride) was added and stirred for 5 minutes togive the symmetrical anhydride of HBB, which was then coupled toAoc-Glu(OBzl)-Trp-Aib-Lys(N.sup.ε -2ClZ)-pMBHA in 10× excess per each oftwo couplings. The HBB-Aoc-Glu(OBzl)-Trp-Aib-Lys (N.sup.ε -2ClZ)-pMBHAprotected peptide was cleaved from the resin and side chain protectinggroups were removed by treating the HBB-peptide-resin in anhydrous HFcontaining 5% anisole and 5% dimethylsulfide at -5° C. for 1 hour. Thepeptide was then extracted from the resin with 50% acetonitrile in 0.1%trifluoroacetic acid, frozen and lypohilized. The peptide was thenpurified by reverse phase HPLC to give the title compound.

1(B). Preparation of Dppc Complex with Polypeptide Described in Example1(A)

Peptide 1(A) is prepared as described above. DPPC (25 mg) in 1 ml ofchloroform is dried under a stream of nitrogen and dried under vacuum toremove traces of organic solvent. To the dry lipid mixture is added 3 mlof water. The preparation is incubated for 1 hour at 45° C. Then, 0.5 mgof dry peptide 1(A) is added to the aqueous preparation. The preparationis sonicated in a bath ultrasonicator at 45° C. for 2 hours. Theresulting lipid-peptide mixture is lyophilized and stored at 4° C. forup to one month. Prior to testing, 9 ml of 0.9% NaCl, 20 mM HEPESbuffer, pH 7.40 is added. The preparation is incubated for 1 hour at 45°C. with periodic mixing.

EXAMPLE 2

2(A). Preparation of Polypeptide HBB-Aoc-Glu-Trp-Glu-Lys-NH₂ (SEQ ID NO:2) (HBB-Aoc=N.sup.α -hydroxy-di-t-butyl-benzoyl-aminoocatanoyl-)

Aoc-Glu(OBzl)-Trp-Glu(OBzl)-Lys(N.sup.ε -2ClZ)-pMBHA was prepared byusing a Lys(N.sup.ε -2ClZ)-pMBHA resin placed in ABI430A peptidesynthesizer and synthesized using standard t-Boc chemistry. Tosynthesize peptide 2A, N.sup.α -hydroxy-di-t-butyl-benzoic acid (HBB)(501 mg), dimethylformamide (4 mL) and methylene chloride (4 mL) werecombined and a dicyclohexylcarbodiimide solution (8 mL of a 0.5Msolution in methylene chloride) was added and stirred for 5 minutes togive the symmetrical anhydride of HBB, which was then coupled toAoc-Glu(OBzl)-Trp-Glu(OBzl)-Lys(N.sup.ε -2ClZ)-pMBHA in 4× excess pereach of two couplings. The HBB-Aoc-Glu(OBzl)-Trp-Glu(OBzl)-Lys(N.sup.ε-2ClZ)-pMBHA protected peptide was cleaved from the resin and side chainprotecting groups were removed by treating the HBB-peptide-resin inanhydrous HF containing 5% anisole and 5% dimethylsulfide at -5° C. for1 hour. The peptide was then extracted from the resin with 50%acetonitrile in 0.1% trifluoroacetic acid, frozen and lypohilized. Thepeptide was then purified by reverse phase HPLC to give the titlecompound.

2(B). Preparation of Dppc Complex with Polypeptide Described in Example2(A)

Peptide 2(A) was mixed with DPPC essentially as described under Example1.

EXAMPLE 3

3(A). Preparation of Polypeptide (Trl-Aoc-Glu-Trp-Aib-Lys-NH₂(Trl-Aoc-=Nα-hydroxy-di-t-butyl-benzoyl-aminoocatanoyl) (SEQ ID NO: 3)

Aoc-Glu(OBzl)-Trp-Aib-Lys(N.sup.ε -2ClZ)-pMBHA was prepared by using aLys(N.sup.ε -2ClZ)-pMBHA resin placed in ABI430A peptide synthesizerusing standard t-Boc chemistry.

To synthesize peptide 3A,6-hydroxy-2,5,7,8,-tetramethylchroman-2-carboxylic acid (Trolox) (501mg), dimethylformamide (4 mL) and methylene chloride (2.5 mL) werecombined and a dicyclohexylcarbodiimide solution (8 mL) of a 0.5Msolution in methylene chloride) was added and stirred for 5 minutes togive the symmetrical anhydride which was then coupled toAoc-Glu(OBzl)-Trp-Aib-Lys(N.sup.ε -2ClZ)-pMBHA in 10× excess per each oftwo couplings.

To cleave Trl-Aoc-Glu(OBzl)-Trp-Aib-Lys(N.sup.ε -2ClZ)-pMBHA from theresin and remove side chain protecting groups, the peptide was treatedwith anyhdrous HF, 5% anisole and 5% dimethylsulfide at -5° C. for 1hour. The Trl-peptide was extracted from the resin with 50% acetonitrilein 0.1% trifluoroacetic acid, frozen and lypohilized. The Trl-peptidewas purified by reverse phase HPLC to give the title compound.

3(B). Preparation of Dppc Complex with Polypeptide Described in Example3(A).

Peptide 3(a) was prepared with DPPC essentially as described in Example1b.

EXAMPLE 4

4(A). Preparation of Polypeptide HBB-Glu-Trp-Aib-Lys-NH₂ (SEQ ID NO: 4)(HBB=N.sup.α -hydroxy-di-t-butyl-benzoyl)

Peptide 4(A) is prepared in a manner essentially analogous to thepreparation of peptide 1(A).

4(B). Preparation of Dppc Complex with Polypeptide Described in Example4(A)

Peptide 4(A) is mixed with DPPC essentially as described under Example1.

EXAMPLE 5

5(A). Preparation of Polypeptide HBB-Aoc-Glu-Trp-Ala-Lys-NH₂ (SEQ ID NO:5) (HBB-Aoc=N.sup.α -hydroxy-di-t-butyl-benzoyl-aminoocatanoyl-)

Peptide 5(A) is prepared in a manner essentially analogous to thepreparation of peptide 1(A).

5(B). Preparation of Dppc Complex with Polypeptide Described in Example4(A).

Peptide 5(A) is mixed with DPPC essentially as described under Example1.

                                      TABLE 1    __________________________________________________________________________    ANALYTICAL PROPERTIES OF PEPTIDES SYNTHESIZED    FABS-MASS SPECTROMETRY ANALYSIS OF PEPTIDES 1-7    SEQ ID    No:  PEPTIDE            FAB MS     AAA    __________________________________________________________________________    1    HBB--Aoc--Glu--Trp--Aib--Lys--NH.sub.2                             M + H!.sup.+  = 920.6                                       @85%    2    HBB--Aoc--Glu--Trp--Glu--Lys--NH.sub.2                             M + H!.sup.+  = 963.6                                       @62%    3    Trl--Aoc--Glu--Trp--Aib--Lys--NH.sub.2                             M + H!.sup.+  = 920.6                                       @89%    4    HBB--Glu--Trp--Aib--Lys--NH.sub.2                             M + H!.sup.+  = 778.97                                       @78%    5    HBB--Aoc--Glu--Trp--Ala--Lys--NH.sub.2                             M + H!.sup.+  = 904                                       @76%    __________________________________________________________________________

Preparation of Antioxidant Moieties

The following antioxidant starting materials may be used as described inthe preceding examples.

EXAMPLE 6 Preparation of Starting Material Aantioxidant Compound3-t-Butyl-5-methyl-4-hydroxybenzoic acid

Charge a reaction vessel with a suspension of sodium hydride (4.74 g,0.198 mol) in anhydrous ethylene glycol dimethyl ether (150 mL). Add, bydropwise addition, a solution of 2-t-butyl-6-methylphenol (0.1 mol) inethylene glycol dimethyl ether (150 mL). Warm to 50°-60° C. for 1.5hours then introduce carbon dioxide through a gas-disparging tube belowthe surface of the reaction mixture for 20 hours. Cool to 5° C. anddestroy the excess sodium hydride carefully with methyl alcohol (30 mL).After hydrogen evolution ceases, adjust the pH of the reaction mixtureto 2 with 1N hydrochloric acid. Dilute with water (1.6 L) and collectthe title compound by filtration.

EXAMPLE 7 Preparation of Starting Material Antioxidant Compound(6-Hydroxy-7-t-butyl-5-isopropyl-8-propylchroman-2-yl)acetic acid

Mix magnesium turnings (45 mg, 1.85 mmol) and1-chloro-2,2-dimethylpropane (74.6 mg, 0.7 mmol) in anhydrous ether (9mL). Heat and stir vigorously, then add, by dropwise addition,1,2-dibromoethane (156 mg, 0.839 mmol) in anhydrous ether (1.5 mL).Reflux for 12 hours, place under an argon atmosphere and cool to 0°-5°C. Add, by dropwise addition, a solution of isobutyryl chloride (0.533mmol) in anhydrous diethyl ether (1.5 mL). Stir at 0°-5° C. for 1.5hours, pour into a mixture of ice and concentrated hydrochloric acid(0.15 mL) and separate the organic phase. Wash with ethyl acetate, 5%aqueous sodium carbonate and brine. Dry (MgSO₄) and evaporate thesolvent invacuo to give 2,2-6-trimethyl-4-heptanone.

Dissolve vinylmagnesium chloride (0.7 mmol) in anhydrous diethyl ether(1 mL), place under an argon atmosphere and cool to 1°-5° C. Add, bydropwise addition, a solution of butyryl chloride (0.533 mmol) inanhydrous diethyl ether (1.5 mL). Stir at 0°-5° C. for 1.5 hours, pourinto a mixture of ice and concentrated hydrochloric acid (0.15 mL) andseparate the organic phase. Wash with water, 5% aqueous sodium carbonateand brine. Dry (MgSO₄) and evaporate the solvent invacuo to give propylvinyl ketone.

Dissolve 2,2-6-trimethyl-4-heptanone (0.4 mol) in methanol (10 mL) andadd potassium tert-butoxide (12 g. 0.1 mol). Add, by dropwise addition,a solution of propyl vinyl ketone (0.2 mol) in methanol (10 mL). Stirfor 10 minutes and portion between ethyl ether and brine. Separate theorganic phase and wash with brine until neutral. Dry (Na₂ SO₄) andevaporate the solvent in vacuo to give2-propyl-3-t-butyl-5-isopropylbenzoquinone.

Dissolve 2-propyl-3-t-butyl-5-isopropylbenzoquinone (10 mmol),1,1,3,3-tetramethyldisiloxane (1.79 mL, 10 mmol) and iodine (0.05 g) inmethylene chloride (30 mL). Stir at reflux for 30 minutes and extractwith 1N sodium hydroxide (30 mL). Acidify the aqueous phase withconcentrated hydrochloric acid and extract into ethyl acetate (4×10 mL),dry (Na₂ SO₄) and evaporate the solvent in vacuo to give2-propyl-3-t-butyl-4-hydroxy-5-isopropylphenol.

Dissolve 2-propyl-3-t-butyl-4-hydroxy-5-isopropylphenol (2.0 mol) andtrimethyl orthoformate (0.3 L) in methanol (1.2 L) and degas. Placeunder a nitrogen atmosphere and cool to 3° C. and add concentratedsulfuric acid (5 mL). Add, by dropwise addition, methyl vinyl ketone(340 mL, 4.0 mol) and stir without cooling for 44 hours. Pour intoaqueous sodium hydrogen carbonate and extract into ethyl ether. Dry(MgSO₄) and evaporate the solvent invacuo to give2-methoxy-2-methyl-7-t-butyl-5-isopropyl-8-propyl-chroman-6-ol.

Dissolve 2-methoxy-2-methyl-7-t-butyl-5-isopropyl-8-propyl-chroman-6-ol(2 mol) in pyridine (600 mL) and add acetic anhydride (900 mL). Degasand stir under a nitrogen atmosphere for 18 hours. Pour into ice/waterand stir for 3 hours. Extract into ethyl ether, dry (MgSO₄), evaporatethe solvent in vacuo and purify by chromatography to give2-methoxy-2-methyl-7-t-butyl-5-isopropyl-8-propyl-chroman-6-yl-acetate.

Dissolve2-methoxy-2-methyl-7-t-butyl-5-isopropyl-8-propyl-chroman-6-yl-acetate(2 mol) in acetone (2.5 L) and add water (2 L) followed by concentratedhydrochloric acid (16.6 mL). Distill the solvent from the stirredmixture until the head temperature reaches 90° C. Cool the suspension,dilute with ethyl ether and wash with aqueous sodium hydrogen carbonate.Dry (MgSO₄), evaporate the solvent invacuo and purify by chromatographyto give2-hydroxy-2-methyl-7-t-butyl-5-isopropyl-8-propyl-chroman-6-yl-acetate.

Suspend sodium hydride (47.2 g of 56% in mineral oil, 1.10 mol) inanhydrous tetrahydrofuran (1 L). Place under a nitrogen atmosphere andadd, by dropwise addition, trimethyl phosphonoacetate (209.4 g, 1.15mol). Stir the 25 minutes and add a solution of2-hydroxy-2-methyl-7-t-butyl-5-isopropyl-8-propyl-chroman-6-yl-acetate(0.5 mol) in tetrahydrofuran (1 L). Stir at room temperature for 18 hourthen heat at reflux for 4 hours. Cool, evaporate the solvent in vacuoand purify by chromatography to give the title compound.

BIOLOGICAL

Methods of testing the synthetic surfactant preparations for efficacyare well known in the art. For example, the synthetic surfactantpreparations of the present invention can be tested in any appropriatemanner such as in the adult rat lung model (Ikegami, et al., (1979)Pediatr. Res. 13, 777-780).

Pressure-volume characteristics of surfactant-depleted rat lungs aresimilar to those of lungs of infants with hyaline membrane disease andrestoration of the pressure-volume relationship of the lung to normal isrelated to the amount of surfactant instilled in a dose dependentmanner. (Bermel, M. S., et al., Lavaged excised rat lungs as a model ofsurfactant deficiency, Lung 162: 99-113 (1984)).

EXAMPLE 8 Isolated Rat Lavaged Lung Model

The experimental procedures for animal preparation, pressure-volumecurve registration and lung lavage are adapted from those described byIkegami et al., Pediatr. Res. 11: 178-182 (1977) and Pediatr. Res. 13:777-780 (1979, and Bermel et al, Lung 162: 99-113 (1984). Male SpragueDawley rats (200-250 g) are anesthetized with sodium pentobarbital andexsanguinated. The trachea is cannulated and the thoracic organs areremoved en bloc. After removal of the adventitious tissue, the tracheaand lungs (˜2 g) are suspended in saline (0.9%), placed in a vacuumchamber, and degassed according to the procedure of Stengel et al. thedegassed lungs are suspended in saline in a 37° C., jacketed reservoirand the tracheal cannula is connected both to a water manometer and aglass syringe by a T-tube. The glass syringe is placed in aninfusion/withdrawal pump. Lungs are rapidly inflated with air to 30 cmH₂ O pressure at the rate of 10 ml/min to minimize air trapping, and aremaintained at this pressure for 10 min by intermittently adding air tothe lungs. The total volume of air infused is recorded as the total lungcapacity (TLC) which is generally 14-15 ml. The lungs are then deflatedat a rate of 2.5 ml/min until zero pressure is attained. Duringdeflation, pressure is read from the water manometer at 1 cm intervalsand recorded. These data are used to construct a pressure-volume (P-V)or quasi-static compliance curve after correction for the P-V curve ofthe apparatus. After degassing and equilibration, the lungs are renderedsurfactant-deficient by repeated lavage with 5 ml/g lavage buffer (0.9%NaCl, 10 mM HEPES, pH 7.4). The procedures of degassing, equilibrating,and lavaging are repeated (15-20 times) until the pressure-volume curvehad become distinctly sigmoidal in shape and the volume of air remainingin the lungs at 5 cm H₂ O pressure is less than or equal to 3 ml. Atthis point, the lungs are considered surfactant-deficient. For testing,2 ml of 0.9% NaCl, 10 mM HEPES buffer, pH 7.4, are added to the dry lungsurfactants (25 mg of phospholipid; 100-125 mg/kg) and the mixture isvortexed, flushed with nitrogen and incubated for 1 h at 45° C. Themixture is then vortexed again, degassed if foamy, and 2 ml of the testmixture are introduced into and withdrawn from the lungs four times bysyringe. When the test mixture is reintroduced to the lungs for thefifth time, it is allowed to remain in the lungs. This procedure isadopted to encourage even distribution of the material throughout thelung. The lungs are degassed, allowed to equilibrate at 37° C. for 5min, and a P-V measurement is performed. Lungs are studied whilesupported in saline at 37° C. as opposed to ambient temperature sincethe physical characteristics of the surfactants may be dependent upontemperature. Canine lung surfactant is administered in a similar mannerexcept that the surfactant is heated for only 5 min. Data are presentedin terms of the % TLC. The deflation limbs of the pressure-volume (P-V)curves in adult rat lungs are analyzed by calculating the total lungcapacities (% TLC) at 5 and 10 cm H₂ O pressure (PC₅ and PC₁₀).Comparisons are based on per cent restoration=(PC₅(sufficient) -PC₅(test) ×100/(PC₅ (sufficient) -PC₅(deficient)) and made by one-wayanalysis of variance using the general linear models procedure withspecific contrasts of the means (SAS Institute Inc., Cary, N.C.). Lavageand treatment with test mixtures did not produce a change in theabsolute TLC of greater than 6%.

Results

The preparations administered to the rat had a translucent appearance.The deflation limb of the pressure-volume (P-V) curve in adult rat lungswas analyzed by calculation of the per cent of total lung capacity (TLC)at 5 cm H₂ O pressure (PC₅) and the TLC at 10 cm H₂ O (PC₁₀). Therestoration based upon the PC₅ values was used to compare the testmixtures. DPPC alone had no significant effect on the pressure-volume(P-V) curves of the lavaged lung. Activities of peptide-DPPC mixturesare indicated in Table 2.

                                      TABLE 2    __________________________________________________________________________    Efficacy of Synthetic Surfactants in The Adult Rat Lavaged Lung Model                         PC.sub.5                              PC.sub.10                                   RESTORATION    Mixture            n (% TLC)                              (% TLC)                                   %    __________________________________________________________________________    sufficient         50                         68 ± 1                              87 ± 1                                   100    deficient          50                         17 ± 1                              45 ± 1                                    0    DPPC                4                         13 ± 1                              31 ± 2                                   11 ± 8    SEQ ID No: 1        2                         48 ± 5                              73 ± 3                                   65 ± 5    HBB--Aoc--Glu--Trp--Aib--Lys--NH.sub.2    SEQ ID No: 2        3                         52 ± 2                              75 ± 2                                   83 ± 5    HBB--Aoc--Glu--Trp--Glu--Lys--NH.sub.2    SEQ ID NO: 3        2                         33 ± 2                              59 ± 2                                   43 ± 6    Trl--Aoc--Glu--Trp--Aib--Lys--NH.sub.2    SEQ ID NO: 5        3                         55 ± 4                              77 ± 2                                   81 ± 7    HBB--Aoc--Glu--Trp--Ala--Lys--NH.sub.2    __________________________________________________________________________

    __________________________________________________________________________    SEQUENCE LISTING    (1) GENERAL INFORMATION:    (iii) NUMBER OF SEQUENCES: 5    (2) INFORMATION FOR SEQ ID NO:1:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 4 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (ix) FEATURE:    (A) NAME/KEY: Modified-site    (B) LOCATION: 1    (D) OTHER INFORMATION: /note=    "Xaa=N-alpha- N-(8-hydroxy-di-t-butyl-benzoyl)-am    ino octanoic!-glutamic acid (HBB-Aoc-Glu)"    (ix) FEATURE:    (A) NAME/KEY: Modified-site    (B) LOCATION: 3    (D) OTHER INFORMATION: /note= "Xaa=2-amino-isobutyric acid    (Aib)"    (ix) FEATURE:    (A) NAME/KEY: Modified-site    (B) LOCATION: 4    (D) OTHER INFORMATION: /note= "Xaa=lysin-1-amide"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:    XaaTrpXaaXaa    (2) INFORMATION FOR SEQ ID NO:2:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 4 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (ix) FEATURE:    (A) NAME/KEY: Modified-site    (B) LOCATION: 1    (D) OTHER INFORMATION: /note=    "Xaa=N-alpha- N-(8-hydroxy-di-t-butyl-benzoyl)-am    ino octanoic!-glutamic acid (HBB-Aoc-Glu)"    (ix) FEATURE:    (A) NAME/KEY: Modified-site    (B) LOCATION: 4    (D) OTHER INFORMATION: /note= "Xaa=lysin-1-amide"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:    XaaTrpGluXaa    1    (2) INFORMATION FOR SEQ ID NO:3:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 5 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (ix) FEATURE:    (A) NAME/KEY: Modified-site    (B) LOCATION: 1    (D) OTHER INFORMATION: /note=    "Xaa=N-alpha- N-(6-hydroxy-2,5,7,8-tetramethyl-ch    roman-2- carboxylic acid)-amino octanoic!"    (ix) FEATURE:    (A) NAME/KEY: Modified-site    (B) LOCATION: 1    (D) OTHER INFORMATION: /note= "(cont'd) -glutamic acid    (Trl-Aoc-Glu)"    (ix) FEATURE:    (A) NAME/KEY: Modified-site    (B) LOCATION: 4    (D) OTHER INFORMATION: /note= "Xaa=2-amino-isobutyric acid    (Aib)"    (ix) FEATURE:    (A) NAME/KEY: Modified-site    (B) LOCATION: 5    (D) OTHER INFORMATION: /note= "Xaa=lysin-1-amide"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:    XaaGluTrpXaaXaa    15    (2) INFORMATION FOR SEQ ID NO:4:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 4 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (ix) FEATURE:    (A) NAME/KEY: Modified-site    (B) LOCATION: 1    (D) OTHER INFORMATION: /note=    "Xaa=N-alpha- N-(8-hydroxy-di-t-butyl-benzoyl)-gl    utamic acid (HBB-G..."    (ix) FEATURE:    (A) NAME/KEY: Modified-site    (B) LOCATION: 3    (D) OTHER INFORMATION: /note= "Xaa=2-amino-isobutyric acid    (Aib)"    (ix) FEATURE:    (A) NAME/KEY: Modified-site    (B) LOCATION: 4    (D) OTHER INFORMATION: /note= "Xaa=lysin-1-amide"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:    XaaTrpXaaXaa    1    (2) INFORMATION FOR SEQ ID NO:5:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 4 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (ix) FEATURE:    (A) NAME/KEY: Modified-site    (B) LOCATION: 1    (D) OTHER INFORMATION: /note=    "Xaa=N-alpha- N-(8-hydroxy-di-t-butyl-benzoyl)-am    ino octanoic!-glutamic acid (HBB-Aoc-Glu)"    (ix) FEATURE:    (A) NAME/KEY: Modified-site    (B) LOCATION: 4    (D) OTHER INFORMATION: /note= "Xaa=lysin-1-amide"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:    XaaTrpAlaXaa    1    __________________________________________________________________________

What is claimed is:
 1. A complex of a polypeptide of the formula:

    X--A.sub.1 --A.sub.2 --A.sub.3 --A.sub.4 --Y

or an optically active isomer or pharmaceutically acceptable saltthereof; wherein A₁ is a bond or negatively charged amino acid selectedfrom Glu or Asp; A₂ is a hydrophobic amino acid selected from Trp, Tyr,Phe, His, Val, Leu, or Ile; A₃ is Aib, Glu, Gln, Leu, Ala, Orn or abond; and A4 is a positive charged amino acid selected from Lys, Arg, orHis; X is of formula Da or Db: ##STR4## wherein, B₁ is B, --C(O)--,--B--C(O)--, --C(O)--NH--B--C(O)--; and B is a bond, C₁₋₁₆ alkylene, orC₂₋₁₆ alkenylene; and wherein each R₁, R₂, R₃, R₄, R₅, R₆ and R₇ isindependently a C₁₋₆ alkyl; Y is a carboxyl substituent of A₄ selectedfrom hydroxy, amino, alkylamino, and alkoxy groups; and wherein, when A₃is a bond, A₁ and A₂ may be interchanged; and a lipid or mixture oflipids selected from the group consisting of DPPC, PC, CL, PG, PS, FAand TG.
 2. A complex of a polypeptide of the formula:

    X--A.sub.1 --A.sub.2 --A.sub.3 --A.sub.4 --Y

or an optically active isomer or pharmaceutically acceptable saltthereof; wherein A₁ is a bond or negatively charged amino acid selectedfrom Glu or Asp; A₂ is a hydrophobic amino acid selected from Trp, Tyr,Phe, His, Val, Leu, or Ile; A₃ is Aib, Glu, Gln, Leu, Ala, Orn or abond; and A₄ is a positive charged amino acid selected from Lys, Arg, orHis; X is of formula Da or Db: ##STR5## wherein, B₁ is B, --C(O)--,--B--C(O)--,--C(O)--NH--B--C(O)--; and B is a bond, C₁₋₁₆ alkylene, orC₂₋₁₆ alkenylene; and wherein each R₁, R₂, R₃, R₄, R₅, R₆ and R₇ isindependently a C₁₋₆ alkyl; Y is a carboxyl substituent of A₄ selectedfrom hydroxy, amino, alkylamino, and alkoxy groups; and a lipid ormixture of lipids selected from the group consisting of DPPC, PC, CL,PG, PS, FA and TG.
 3. A complex as in claim 1 or 2 in which DPPCcomprises the major component of the lipid.
 4. A complex as in claim 1or 2 in which the lipid is a mixture of DPPC and PG.
 5. A complex as inclaim 1 or 2 in which the lipid consists of from about 85-100% DPPC andfrom about 0-15% PG.
 6. A complex as in claim 1 or 2 in which thepolypeptide is HBB-Aoc-Glu-Trp-Aib-Lys-NH₂ (SEQ ID NO: 1).
 7. A complexas in claim 1 or 2 in which the polypeptide isHBB-Aoc-Glu-Trp-Glu-Lys-NH₂ (SEQ ID NO: 2).
 8. A complex as in claim 1or 2 in which the polypeptide is Trl-Aoc-Glu-Trp-Aib-Lys-NH₂ (SEQ ID NO:3).
 9. A complex as in claim 1 or 2 in which the polypeptide isHBB-Glu-Trp-Aib-Lys-NH₂ (SEQ ID NO: 4).
 10. A complex as in claim 1 or 2in which the polypeptide is HBB-Aoc-Glu-Trp-Ala-Lys-NH₂ (SEQ ID NO: 5).11. A method of treating respiratory distress syndrome in a subject inneed thereof which comprises administering to the subject an effectiveamount of a complex of a polypeptide of the formula:

    X--A.sub.1 --A.sub.2 --A.sub.3 --A.sub.4 --Y

or an optically active isomer or pharmaceutically acceptable saltthereof; wherein A₁ is a bond or negatively charged amino acid selectedfrom Glu or Asp; A₂ is a hydrophobic amino acid selected from Trp, Tyr,Phe, His, Val, Leu, or Ile; A₃ is Aib, Glu, Gln, Leu, Ala, Orn or abond; and A₄ is a positive charged amino acid selected from Lys, Arg, orHis; X is of formula Da or Db: ##STR6## wherein, B₁ is B, --C(O)--,--B--C(O)--,--C(O)--NH--B--C(O)--; and B is a bond, C₁₋₁₆ alkylene, orC₂₋₁₆ alkenylene; and wherein each R₁, R₂, R₃, R₄, R₅, R₆ and R₇ isindependently a C₁₋₆ alkyl; Y is a carboxyl substituent of A₄ selectedfrom hydroxy, amino, alkylamino, and alkoxy groups; and wherein, when A₃is a bond, A₁ and A₂ may be interchanged; and a lipid or mixture oflipids selected from the group consisting of DPPC, PC, CL, PG, PS, FAand TG.
 12. A method of treating respiratory distress syndrome in asubject in need thereof which comprises administering to the subject aneffective amount of a complex of a polypeptide of the formula:

    X--A.sub.1 --A.sub.2 --A.sub.3 --A.sub.4 --Y

or an optically active isomer or pharmaceutically acceptable saltthereof; wherein A₁ is a bond or negatively charged amino acid selectedfrom Glu or Asp; A₂ is a hydrophobic amino acid selected from Trp, Tyr,Phe, His, Val, Leu, or Ile; A₃ is Aib, Glu, Gln, Leu, Ala, Orn or abond; and A₄ is a positive charged amino acid selected from Lys, Arg, orHis; X is of formula Da or Db: ##STR7## wherein, B₁ is B, --C(O)--,--B--C(O)--,--C(O)--NH--B--C(O)--; and B is a bond, C₁₋₁₆ alkylene, orC₂₋₁₆ alkenylene; and wherein each R₁, R₂, R₃, R₄, R₅, R₆ and R₇ isindependently a C₁₋₆ alkyl; Y is a carboxyl substituent of A₄ selectedfrom hydroxy, amino, alkylamino, and alkoxy groups; and a lipid ormixture of lipids selected from the group consisting of DPPC, PC, CL,PG, PS, FA and TG.
 13. A method as in claim 11 or 12 in which DPPCcomprises the major component of the lipid.
 14. A method as in claim 11or 12 in which the lipid is a mixture of DPPC and PG.
 15. A method as inclaim 11 or 12 in which the lipid consists of from about 85-100% DPPCand from about 0-15% PG.
 16. A method as in claim 11 or 12 in which thepolypeptide is HBB-Aoc-Glu-Trp-Aib-Lys-NH₂ (SEQ ID NO: 1).
 17. A methodas in claim 11 or 12 in which the polypeptide isHBB-Aoc-Glu-Trp-Glu-Lys-NH₂ (SEQ ID NO: 2).
 18. A method as in claim 11or 12 in which the polypeptide is Trl-Aoc-Glu-Trp-Aib-Lys-NH₂ (SEQ IDNO: 3).
 19. A method as in claim 11 or 12 in which the polypeptide isHBB-Glu-Trp-Aib-Lys-NH₂ (SEQ ID NO: 4).
 20. A method as in claim 11 or12 in which the polypeptide is HBB-Aoc-Glu-Trp-Ala-Lys-NH₂ (SEQ ID NO:5).